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Shape-dependent bactericidal activity of copper oxide nanoparticle
mediated by DNA and membrane damage
Dipranjan Laha a, Arindam Pramanik a, Aparna Laskar c, Madhurya Jana a,
Panchanan Pramanik b, Parimal Karmakar a,*
a Department of Life Science and Biotechnology, Jadavpur University, 188, Raja S C Mallick Road, Kolkata 700032, India
b Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India
c CSIR-Indian Institute of Chemical Biology, Kolkata 700032, India
A R T I C L E I N F O
Article history:
Received 7 May 2014
Received in revised form 14 June 2014
Accepted 22 June 2014
Available online 10 July 2014
Keywords:
B. Chemical synthesis
A. Metals
C. Atomic force microscopy
A B S T R A C T
In this work, we synthesized spherical and sheet shaped copper oxide nanoparticles and their physical
characterizations were done by the X-ray diffraction, fourier transform infrared spectroscopy,
transmission electron microscopy and dynamic light scattering. The antibacterial activity of these
nanoparticles was determined on both gram positive and gram negative bacterial. Spherical shaped
copper oxide nanoparticles showed more antibacterial property on gram positive bacteria where as sheet
shaped copper oxide nanoparticles are more active on gram negative bacteria. We also demonstrated that
copper oxide nanoparticles produced reactive oxygen species in both gram negative and gram positive
bacteria. Furthermore, they induced membrane damage as determined by atomic force microscopy and
scanning electron microscopy. Thus production of and membrane damage are major mechanisms of the
bactericidal activity of these copper oxide nanoparticles. Finally it was concluded that antibacterial
activity of nanoparticles depend on physicochemical properties of copper oxide nanoparticles and
bacterial strain.
ã 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Nanostructured materials offer promising opportunities for
improved applications in different area of modern life due to their
unique physicochemical properties, caused by their nanosized
dimensions and large surface/volume ratios [1]. More recently,
several natural and engineered nanomaterials have been shown to
possess strong antimicrobial properties including silver nano-
particles [2], TiO2 [3], ZnO [4] and SiO2 [5]. Some nanocomposite
consisting of different materials are possess bactericidal activity.
For example, microfibril bundles of cellulose substance with
titania/chitosan/silver-nanoparticle composite films and hierar-
chical nanofibrous titania–carbon composite material deposited
with silver nanoparticles are lethal to various bacterial strains
[6,7].
Application of antibacterial agents in the textile industry, water
disinfection, medicine, food packaging etc. are well known. Unlike
conventional chemical disinfectants, the antimicrobial nanomate-
rials are not expected to produce harmful disinfection by products
(DBPs). Among these several metal based nanoparticles (e.g., copper
based nanoparticle) are increasingly recognized as a suitable
alternative due to its high redox potential property and relatively
lowercostofproduction[8]. Previously, ithas beenreportedthatCuO
NPs exibit strong antimicrobial activity against broad spectrum of
gram positive and gram negative bacteria [9]. Though, the constit-
uents of cell wall in gram-positive and gram-negative bacteria are
mainly responsible for their sensitivity to CuO NPs but other factors
can also influence the sensitivity. For instance, gram negative E (!) is
highlysensitive,but S.aureus(+)andB.subtilis(+)arelesssensitiveto
CuO NPs [8]. On the other hand bactericidal activity of such
nanoparticles in part depends on size, stability, shape and
concentration in the growth medium [10,11].
The mechanisms by which such metal oxide nanoparticles
induce bactericidal activities is not fully known but amount of ion
release and subsequent production of ROS is supposed to be the
main cause [12]. The rate of dissolution of such nanoparticles
depends on their morphology as well as their nature [13].
Additionally, by electrostatic interaction nanoparticles are able
to attach to the membrane of bacteria and interfere with bacterial
membrane [14]. Depending on these two factors many metal oxide
nanoparticles act differentially on different strain. As the way by
which bacteria is killed by such nanoparticles is different from the
* Corresponding author. Tel.: +91 3324146710; fax: +91 3324137121.
E-mail address: pkarmakar_28@yahoo.co.in (P. Karmakar).
http://dx.doi.org/10.1016/j.materresbull.2014.06.024
0025-5408/ã 2014 Elsevier Ltd. All rights reserved.
Materials Research Bulletin 59 (2014) 185–191
Contents lists available at ScienceDirect
Materials Research Bulletin
journal homepage: www.else vie r.com/locat e/mat resbu
antibiotic their proper evaluation is necessary. Thus a comprehen-
sive knowledge about their size and morphology depended anti
bacterial activity must be evaluated. In light of these, we undertook
the effort to assess the morphology dependent activity of CuO NPs
on different bacterial strain. We have synthesized two different
shapes of CuO NPs and characterized them for their antimicrobial
activity. The antibacterial activity was examined on a broad range
of bacterial species including E.scherichia coli wild type, Micrococ-
cus luteus, Bacillus subtilis and Proteus vulgaris. While sheet shape
CuO NPs are potentially active against gram positive bacteria and
spherical shaped CuO NPs are more effective on gram negative
bacteria. Both membrane damage and ROS mediated DNA damage
are responsible for their antimicrobial activity.
2. Materials and methods
2.1. Materials
In this study all chemicals of analytical grade were used. Copper
acetate [Cu(CH3COO)2], glacial acetic acid [CH3COOH], sodium
hydroxide [NaOH], copper nitrate trihydrate [Cu(NO3)2″3H2O] was
obtained from SRL, India, ethanol (99%), sodium acetate [CH3COONa]
from Qualigen, India. Alizarin red S (ARS), Hanks balanced salt
solution (HBSS), nitroblue tetrazolium (NBT) were obtained from
Sigma–Aldrich, USA. Hydrochloric acid (35%), dimethyl sulfoxide
(DMSO), Muller–Hinton agar (MHA) medium and Muller–Hinton
broth (MHB) medium were obtained from Hi-media, India.
2.2. Synthesis of CuO NPs (nanospherical, nanosheet)
Different shaped CuO NPs were prepared using co-precipitation
method where either copper acetate or copper nitrate is used to
form CuO NPs and NaOH acts as stabilizing compound [15,16].
2.2.1. Synthesis of CuO nanospherical
300 ml of 0.02 M copper acetate was taken in a conical flask.
1 ml of glacial acetic acid was added to it. The solution is heated at
80–90 #C on a hot plate with vigorous stirring for 10 min by a
magnetic stirrer. 0.8 g NaOH was added rapidly to maintain the pH
6–7. The mixer was kept for 1 h in stirring condition. The resultant
solution was centrifuged at 8000 rpm for 10 min. Pellet was dried
at 37 #C for 3 days. After that it was homogenized by pestle–mortar
and stored.
2.2.2. Synthesis of CuO nanosheet
80 ml of 0.02 M copper nitrate was slowly added to 5 M NaOH
solution in a conical flask at 82 #C. Additional 80 ml of same copper
nitrate solution was added to above solution, a total of 32 g of NaOH
pellet was added to the flask reactor to maintain the constant
concentration of NaOH. The resultant solution was centrifuged at
8000 rpm for 10 min. The pellet was collected and washed with
water. Pellet was dried at 37 #C for 3 days. After that it was
homogenized by pestle–mortar and stored.
2.3. Particle characterization
Thephaseformationandcrystallographicstateofdifferentshaped
CuO NPs were determined by XRD with an Expert Pro (Phillips) X-ray
diffractometer using CoKa radiation (a = 0.178897 nm). Samples
were scanned from 20# to 80# of 2u increment of 0.04# with 2 s
counting time. Presence of surface functional groups was investi-
gated by FTIR spectroscopy (Thermo 132 Nicolet Nexus FTIR, model
870). The particle size and nanostructure were studied by high-
resolution transmission electron microscopy in a JEOL 3010
(HRTEM), Japan operating at 200 KeV. Dry powder of particles was
suspended in de-ionized water at a concentration of 1 mg/mL and
then sonicated at room temperature for 10 min at 40 W to form a
homogeneous suspension. After sonication and stabilization, the
samples were prepared by coating on carbon-coated copper grids
and air dried before TEM analysis. The hydrodynamic size of
dispersed CuO NPs in aqueous phase was measured in a Brookhaven
90 Plus particle size analyzer. Copper based nanoparticles were
dispersed in water to form diluted suspension of 0.5 mg/ml using
sonicator for 30 min. The particles were analyzed by DLS after they
were completely dispersed in water.
2.4. Bacterial strains and culture conditions
Well characterized cells of B. subtilis (ATCC 6633), M. luteous
(ATCC 9341), E. coli (ATCC 10,536), P. vulgaris (ATCC 13,387), DH5a
(k12) were maintained on MHA. Prior to incubation with NPs, the
bacteria were cultured overnight in 4 ml of MHB in shaker at 37 #C
until the optical density (OD) of the culture reached 1.0 at 600 nm,
which indicates 109 CFU ml!1. The overnight cultures were diluted
to 107 CFU ml!1 with sterile broth.
2.5. Antibacterial assay
Antibacterial activity of different shaped CuO NPs was affirmed
through determination of minimum inhibitory concentration (MIC)
and minimum bactericidal concentrations (MBC) [17,18]. MIC is
defined as the lowest concentration of antimicrobial agent at which
no growth is observedin broth medium. Test tubescontaining 4 ml of
broth was inoculated with overnight cultures of the bacteria and
then various concentrations of different shaped CuO NPs (0 mg/ml–
0.4 mg/ml) were added in each tube. The tubes were left for shaking
at 37 #C for 24 h. Then optical density of each tube was measured at
600 nm for the determination of bacterial growth. To substantiate
antibacterial activity further, MBC was determined by inoculating a
loop of NPs treated bacterial culture on MHA plates and left at 37 #C
for 24 h. MBC is defined as the lowest concentration of NPs where no
growth of bacteria is noted on agar plates. Growth curve was studies
for both gram positive and gram negative bacteria with and without
LD50 dose of these nanoparticles for 8 h.
2.6. Reactive oxygen species (ROS) assay
The production of intracellular reactive oxygen species (ROS)
was measured using the same protocol mentioned in our earlier
publication [19].
2.7. In vitro copper ion release study
Release of copper ion from the adsorbed NPs in nutrient broth
was studied by the metallochromic dye ARS. To each test tube, 4 mg
of different shape CuO NPs (nanospherical, nanosheet) were added
in 1 ml of MHB. Then the test tubes were kept under shaking
condition at 37 #C. Supernatant from each test tube was collected
after 2, 4, 6, 12 and 24 h by centrifugation at 10,000 rpm for 10 min.
Next, to each collected supernatant a 100 ml of ARS was added from
stock (10!5 M) along with sodium acetate buffer to maintain acidic
pH. The solution was kept for 10 min and then optical density (OD)
was measured at 510 nm by UV–vis spectrophotometer. The
intensity of absorption depends on the amount of Cu–ARS complex
which in turn depends on the concentration of Cu2+. The
experiment was carried out three times and reproducible data
were obtained [20].
2.8. DNA damage assay
The effect of different shaped CuO NPs on DNA was observed
inside bacterial cell. Reporter (b-galactosidase) gene expression
186 D. Laha et al. / Materials Research Bulletin 59 (2014) 185–191
assay was performed. They were inoculated on agar plates
containing X-gal and IPTG in the medium and incubated for 12 h
at 37 #C for blue color forming colonies [20].
2.9. Cell morphology study by AFM
The effect of different shaped CuO NPs on bacterial cell
morphology was studies using atomic force microscopy (AFM,
Vecco, USA). Fresh E. coli bacterial culture (OD 0.2) were treated
with LD50 dose of NPs for 3 h and then washed with phosphate
buffered saline (pH 7) for three times and the cells were fixed with
2.5% glutaraldehyde. A drop of diluted cell suspension was placed
on a cover slip and allowed to dry before AFM study [21].
2.10. Cell morphology study by SEM
The effect of different shaped CuO NPs on bacterial cell
morphology was studies using scanning electron microscopy (SEM,
Vecco, USA). Fresh bacterial culture (OD 0.2) were treated with
LD50 dose of different shaped CuO NPs for 3 h and then washed
with phosphate buffered saline (pH 7) for three times and the cells
were fixed with 2.5% glutaraldehyde. A drop of diluted cell
suspension was placed on a cover slip and allowed to vacuum dry
before SEM study [22].
2.11. Data analysis
A Student’s t-test was used to calculate the statistical
significance of changes. In all cases, differences are significant
for p < 0.05. Data analysis was performed using the Origin Pro
v.8 software(Origin Lab).
3. Results and discussions
3.1. DLS and TEM analysis
The hydrodynamic size of different shaped CuO NPs was
measured by DLS. Table 1 summarize their physical characteriza-
tion. The TEM micrograph of different shaped copper oxide is
shown Fig. 1A. From the Fig. 1A, the size of spherical and sheet
shaped CuO NPs were 35 $ 5.6, 257.12 $ 13.6 % 42 $ 5.10, respec-
tively. As seen in the table, the hydrodynamic sizes of the
Table 1
Characterization of the different shaped CuO NPs used in this study morphology primary size hydrodynamic diameter zeta potential pDia (TEM) TEM (nm) DLS (nm).
Morphology Primary size Hydrodynamic diameter Zeta potential pDia
(TEM) TEM (nm) DLS (nm)
CuO spherical 33.20 $ 6.18 235 !27.6 0.305
CuO sheet 257.12 $ 13.6 % 42 $ 5.10 372 !23.1 0.346
a Polydispersity index.
Fig. 1. Physical characterization of different shaped CuO NPs (A) X-ray diffraction patterns of CuO nanosheet and CuO nanospherical; (B) FTIR spectra of of CuO nanosheet and
CuO nanospherical; (C) transmission electron microscopic (TEM) image and dynamic light scattering CuO nanosheet and CuO nanospherical.
D. Laha et al. / Materials Research Bulletin 59 (2014) 185–191 187
synthesized NPs were significantly larger than those indicated by
their TEM images. This is possibly due to the fact that TEM
measures size in the dried state of the sample, where as the DLS
measures the size of the hydrated state of particle.
3.2. X-ray diffraction pattern
We first characterized the purity of CuO NPs by XRD. The XRD
pattern of CuO NPs was compared and interpreted with standard
data of the JCPDS file (JCPDS International Center for Diffraction
Data, 1991). Fig. 1B shows the XRD pattern of two different shaped
CuO NPs, the characteristic peaks at 2u = 32.25#, 33.12#, 35.28#,
48.62#, 53.42#, 58.09#, 65.95#,67.90# and 72.24# which are in
agreement with JCPDS card no. 44-0706.
3.3. Compositional and optical analysis of synthesized different shaped
copper oxide nanoparticles (CuO NPs)
The functional or composition quality of the synthesized
product was analyzed by the FTIR spectroscopy. Fig. 1C shows
the FTIR spectrum in the range of 500–4,000 cm!1. The pure CuO
NPs exhibited strong band at 1640 cm!1, characteristic of the CO
stretch and the broad band around 3440 cm!1, indicates the
presence of !!OH groups (Fig. 1C) for both CuO NPs. Table 1
summarized the physical characteristic of CuO NPs.
3.4. Evaluation of antibacterial properties
The antibacterial activities of these two different shaped CuO
NPs against gram positive and negative bacteria were investi-
gated using E. coli, P vulgaris, B.subtilis and M. luteus as model
organisms. Shape dependent activity of CuO NPs was measured by
determining minimum inhibitory concentration (MIC) and mini-
mal bactericidal concentration (MBC) as shown in Tables 2 and 3,
respectively. The growth of gram negative bacteria P. vulgaris and
E.coli was completely inhibited by spherical CuO NPs at a
concentration of 0.16 mg/ml and 0.20 mg/ml, respectively where
as CuO nanosheet was more active on gram positive bacteria B.
subtilis and M. luteous (0.22 mg/ml and 0.20 mg/ml, respectively).
Significance of each MIC value is also determined. Difference in
dose required for both types of nanoparticles to inhibit the growth
of same bacterial strain is also shown on the last column of
Table 2. From the Table 2, it is seen that for nanosheet the MIC
value is 120–140 ug/ml less than nanoshperical for gram positive
bacteria where as for gram negative bacteria, spherical CuO NPs is
120–80 ug/ml less than nanosheet indicating nanosheet CuO NP
are more effective in gram positive bacteria and spherical CuO NP
is effective in gram negative bacteria. We also determined the
MBC of all bacterial strains after treating them with different
shaped CuO NPs. A comprehensive table, showing MIC and MBC of
different bacterial strain and the ratios of MIC and MBC are
Table 2
MIC value of different shaped CuO NPs on different strain.
Bacterial strain
(106 CFU/ml)
Nanospherical (mg/ml) Nanaospherical (mg/ml) p value Difference doses between nanospherical and nanosheet
B. subtilis (+) 0.22 $ 0.028 0.36 $ 0 Nanosheet > nanospherical
(p < 0.05)
140 mg/ml
M. luteous (+) 0.20 $ 0.010 0.32 $ 0 Nanosheet > nanospherical
(p < 0.01)
120 mg/ml
E. coli (!) 0.28 $ 0.024 0.20 $ 0.05 Nanospherical > nanosheet
(p < 0.01)
80 mg/ml
P. vulgaris (!) 0.28 $ 0.0 0.16 $ 0 Nanospherical > nanosheet
(p < 0.05)
120 mg/ml
Table 3
MBC and MBC/MIC value of different shaped CuO NPs on different strain.
B. subtilis (+ve) M. luteus (+ve) P. vulgaris (!ve) E. coli (!ve)
Sph Sheet Sph Sheet Sph Sheet Sph Sheet
MBC(mg/ml) 0.36 0.24 0.32 0.24 0.36 0.36 0.36 0.32
MBC/MIC 1 1.12 1 1.5 1.28 1.28 1.5 1
Fig. 2. (A,B) Growth curve (optical density) of E. coli, P. vulgaris, B. subtilis, M. luteous treated with respective LD50 dose of CuO nanosheet and CuO nanospherical respectively.
188 D. Laha et al. / Materials Research Bulletin 59 (2014) 185–191
presented in Table 3, For all the cases the ratio of MBC to MIC is 1
or greater than 1 indicating the potential bactericidal activity.
LD50 value of different shaped CuO nanoparticles on different
strain was also determine (data not shown). Fig. 2 represents
growth kinetics of different strain bacteria in the presence of
sheet (Fig 2A) and spherical (Fig. 2B). As seen in the Fig 2 the
growth of CuO nanosheet treated bacteria was inhibited after 10 h
whereas CuO NPs spherical treated bacteria reached a stationary
phase after 10 h of growth. In case of all the four microbial strains,
it was observed that with the increase in time of incubation
beyond 10 h, with different shaped CuO NPs, OD value was
decreased.
To determine the possible mechanism of different shaped
CuO NPs on bacterial strains, we assayed in vitro copper ion
release by ARS. As shown in Fig. 3A, copper ion release from
spherical shaped CuO NPs was less than sheet shaped CuO NPs
at early time point but with increasing time the ion release
became same for both the nanoparticles. One step further, we
assayed ROS for bacterial strain E.coli and B. subtilis after the
treatment of CuO NPs at LD50 dose. In E. coli spherical NPs
produced more ROS compared to sheet but for B. subtillis ROS
production was almost same for both the NPs. To check the DNA
damage induced by NPs we used plasmid based reporter gene
assay. In Fig. 3C, reporter gene b-galactosidase was assayed by
transforming DH5a with the plasmid and followed by NPs
treatment. The amount of blue colonies (due to the hydrolysis of
X-gal by b-galactosidase enzyme) reduced significantly for the
bacterial cells treated with NPs. We also used atomic force
microscope to determine the effect of CuO NPs on E. coli. As seen
in Fig 4, both spherical and sheet CuO NPs attached to bacterial
cell membrane. Finally, we used SEM to visualize any membrane
damage of bacteria. From the SEM image it was observed that
Fig. 3. (A) In vitro copper ion release of these two different shaped CuO NPs. (B) Determination of reactive oxygen species (ROS) of E. coli and B. subtilis in presence of these of
different shaped CuO NPs. (C) Reporter gene (b-galactosidase) assay on nanoparticle treated and mock treated pUC 19 transformed DH5a.
Fig. 4. Atomic force microscopy (AFM) images of different shaped copper oxide nanoparticles treated or mock-treated gram negative E. coli bacterial cells.
D. Laha et al. / Materials Research Bulletin 59 (2014) 185–191 189
spherical shaped produced more membrane damage on E. coli
compared to sheet and sheet shaped induced more membrane
damage on B. subtilis (Fig. 5).
4. Discussion
In this study, we have reported the antibacterial activity of
spherical and sheet shaped CuO NPs. Our results showed that the
antibacterial effect of CuO NPs not only depends on size, but also on
specific morphology and nature of the bacterial strain. Being
transition metal, copper plays an important role in cellular redox
cycling and antibacterial activity of copper based NPs are reported
earlier [23,24]. Here we showed that apart from its size, CuO NPs
morphology is also important for antibacterial activity. Previously
Marsili et al. reported morphology dependent antibacterial activity
of zinc oxide nanoparticles [25]. In our case we observed
differential antibacterial activity of rod and spherical shaped
CuO NPs. However, the mechanism of bactericidal actions of these
nanoparticles are still not well understood, but it was proposed
that surface charge of free metal surface is responsible for the
interaction with the bacterial membrane [26]. As a matter of fact
nanoparticles may associate with bacteria through several types of
interaction such as hydrophobic, electrostatic or van der Waals
interaction which may help to damage the cell membrane [27]. In a
previous report it was shown that the interaction between silver
nanoparticles and constituents of the bacterial membrane caused
structural changes in membranes and finally leading to cell death
[28]. Similarly surface modification of gold nanoparticles with BSA
has been shown to determine its biological effects [29].
We observed that gram positive bacteria are more sensitive to
nanosheet CuO NPs where as gram negative are more sensitive to
spherical CuO NPs. It may be due to the fact that large sheet shaped
CuO NPs can not penetrate the outer membrane of gram negative
bacteria, where as small spherical shaped CuO NPs easily penetrate
inside the bacterial cell. On the other hand having more surface
charge, sheet shaped CuO NPs induced more damage to gram
positive bacteria. Such large surface area of diethylaminoethyl
dextran chloride (DEAE-D) functionalized gold nanoparticles also
shown to induce hemolysis in RBC [29].
Previously, several studies reported that two possible mecha-
nisms are involved in the toxicity of nanoparticles on bacterial cell
(1) production of increased level of ROS mostly hydroxyl radicals
and singlet oxygen (2) deposition of nanoparticle on the surface of
bacteria, resulting accumulation of nanoparticles either in the
cytoplasm or in the periplasmic region causing disruption of
cellular function. We have also seen the accumulation of CuO NPs
on bacterial cell surface by AFM. The differential activity of these
two shaped nanoparticles may be due to their difference in ROS
generation inside the cells. In vitro Cu ion release is almost same at
the higher time for both shaped CuO NPs and the amount of ROS
generation is also same by two CuO NPs in B. subtilis strain. It is
likely that sheet shaped NPs have less access inside the cells but
their accumulation in the membrane or periplasmic region perturb
the structure of membrane of such bacteria. This is also observed in
our SEM studies where more membrane damage are observed in B.
subtilis by nanosheet CuONPs. The thick shield of peptidoglycan
layer or its constituents may thus be the target of sheet shaped
CuONPs where as small size spherical CuO NPs easily permeable to
thin peptidoglycan layer of gram negative bacteria and produce
more ROS inside the cell. As a matter of fact these NPs can locally
change microenvironments near the bacteria and produce ROS or
increase the NPs solubility, which can induce bacterial damage.
Thus both spherical and sheet shaped CuO NPs produce membrane
damage to gram negative or gram positive bacteria, as observed by
SEM. The exact mechanisms of action is not known but it seems
likely that constituent of bacterial cell surface may contribute
largely by interacting with specific nanoparticles. Additionally we
found both the nanoparticles produce DNA damage. Large amounts
of ROS could be generated even when only small amounts of CuO
NPs are incorporated into cells. Nanoparticles can induce ROS
directly, once they are exposed to the acidic environment of
lysosomes or interact with oxidative organelles, such as mito-
chondria. Thus, antibacterial activity of these two CuO NPs may
depend on several factors including physiochemical properties of
Fig. 5. Scanning electronic microscopic image (SEM) of different shaped copper oxide nanoparticles treated or mock-treated gram negative and gram positive E. coli bacterial
cells.
190 D. Laha et al. / Materials Research Bulletin 59 (2014) 185–191
nanoparticles and nature of bacterial surface. Thus the nature of
bacterial strain and the surface properties of CuO NPs (e.g., size,
shape, zeta potential etc.) are responsible for the antibacterial
activity.
5. Conclusion
In this study, we presented the antibacterial activity of two
different shaped CuO NPs on different strain. The particles size and
morphology were characterized by DLS and TEM. Chemical
characterization was done by XRD, FTIR. The studies of antibacte-
rial activity of different shaped CuO NPs showed that the NPs were
effective on variety of gram positive and gram negative bacteria as
well as sheet shaped CuO NPs is more active on gram positive
where as spherical shaped CuO NPs is more active gram negative
bacteria. ROS induced DNA damage and membrane ruptures are
the possible mechanisms of antibacterial activity of both shaped
CuO NPs.
Acknowledgements
The authors would like to acknowledge for financial support for
this research work the Department of Biotechnology, Government
of India (No. BT/PR14661/NNT/28/494/2010). We also express
sincere thanks to Indian Institute of Chemical Biology (IICB),
Kolkata, India for providing the facilities to transmission electron
microscopy and atomic force microscopy.
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Name: ______________________
Bio 351 Homework 2 (10 POINTS): DUE MONDAY AT 11PM ON BLACKBOARD.
Background Information: The delivery of antibiotics, or antimicrobials has been extensively studied. Nanoparticles provide a promising alternative to treating infections due to their small size, and variety of applications in the study of microbial medicine. Your job is apply what you have learned in lecture to something you have not seen before. It is necessary to use the knowledge you have to begin or continue to interpret scientific research articles, particularly in the field of microbiology.
Nanoparticles are transport vehicles that deliver ions (at least in the research article I provided). The article provided used two types of CuO (copper oxide nanoparticles) as an antimicrobial: CuO nanosheets (flat structures that contain copper oxide), vs. CuO nanospheres (spherical structures containing copper oxide). Scientists wanted to determine if there was one structure, or if both were effective as a vehicle in delivering copper ions, and if it is an effective antimicrobial towards E. coli, B. subtilus, P. vulgaris, and M. luteous.
Word limit of maximum 40 words per answer. Be concise with your answers. Do not copy the wording from the article, or your textbook.
1. Before you can begin to interpret figures from the research article attached, you must gather information first by answering questions a-c below. Once you have done this, then questions d & e can be answered.
a. Please indicate which of the bacterial species listed are gram-positive and gram-negative. (1 point)
b. What is the difference between antimicrobials and antibiotics? You can perform a Google search to find the answer. (1 point)
c. What are differences seen in cell wall composition between gram-positive and gram-negative bacteria? Use your textbook and lecture notes. (1 point)
d. In Figure 2, what is the optical density from each of the bacterial species? Over a period of time what does this tell you about bacterial growth from each species when exposed to CuO nanosheet (Panel A), and CuO nanospherical (Panel B)? Are gram-positive and gram-negative bacteria used in this study affected similarly or different? If so, how do you know based on Figure 2? (2 points)
e. One method scientists use to determine if a drug-delivery system is effective is by measuring the amount of reactive oxygen species (ROS) generated. ROS are considered free radicals that can harm cells, and their likelihood for survival. Please interpret Figure 3, Panel A & B. (2 points)
f. What were the findings from Figure 5? What could be occurring at the cell wall for gram-positive and gram-negative bacteria? (2 points)
g. Based on the evidence from Figures 2, 3, and 5., which nanoparticle delivery system was most effective as an antimicrobial? (1 point)
Escherichia coli: a Gram-negative bacterium of the gut microbiome, Part 1
FIGURE 3.1 ■ Escherichia coli: a Gram-negative bacterium of the gut microbiome. The envelope includes the outer membrane; the cell wall and periplasm; and the inner (cell) membrane. Embedded in the membranes is the motor of a flagellum. The cytoplasm includes enzymes, messenger RNA extending out of the nucleoid, and ribosomes. Ribosomes translate the mRNA to make proteins, which are folded by chaperones. The nucleoid contains the chromosomal DNA wrapped around binding proteins. (PDB codes: ribosome, 1GIX, 1GIY; DNA-binding protein, 1P78; RNA polymerase, 1MSW)
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Bacterial Cell Structure: What is seen in Gram-negative & Gram-positive bacteria
Bacteria can be placed in 2 groups based on the thickness and placement of the cell wall
Gram-negative
Gram-positive
Plasma membrane
Absence of a nucleus
DNA is located in the nucleiod region
No histone proteins, but DNA-binding proteins present to keep genomic DNA compact
Plasmids: DNA that is independent of the genome.
Flagellum
Biochemical composition of bacteria
Water
Essential Ions
Needed for enzymatic reactions
Small organic molecules: lipids and sugars
Lipids are almost as abundant as RNA molecules
Found in the cell wall
peptidoglycan
Macromolecules: nucleic acids, proteins, fats, & sugars
Goal: Isolate proteins
Purpose of cell fractionation is to isolate components of choice from a bacterial cell
The first step is cell lysis
EDTA
Sucrose
Lyzozymes
Ultracentrifugation
FIGURE 3.2 ■ Fractionation of Gram-negative cells.
Cell periplasm fills with sucrose, and lysozyme breaks down the cell wall. Dilution in water causes osmotic shock to the outer membrane, and periplasmic proteins leak out. Subsequent centrifugation steps separate the proteins of the periplasm, cytoplasm, and inner and outer membranes. Photo
Source: Lars D. Rennera and Douglas B. Weibel. PNAS 108(15):6264.
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Goal 2:Protein Analysis
FIGURE 3.3 ■ Protein analysis.
A. Gel electrophoresis of total cell proteins compared to outer membrane proteins from cell fractionation. B. Outer membrane proteins are identified by tryptic digest and mass spectrum analysis. The resulting peptide sequence is compared with those predicted from genome data.
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FIGURE 3.3a ■ Protein analysis.
A. Gel electrophoresis of total cell proteins compared to outer membrane proteins from cell fractionation.
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FIGURE 3.3b ■ Protein analysis.
B. Outer membrane proteins are identified by tryptic digest and mass spectrum analysis. The resulting peptide sequence is compared with those predicted from genome data.
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Understanding the role of a protein
FIGURE 3.4 ■ Genetic analysis of FtsZ.
A. E. coli with aspartate (D) at position 45 replaced by alanine (A) (D45A) elongate abnormally, forming blebs from the side, with no Z-rings. Cells with aspartate replaced by alanine at position 212 (D212A) elongate to form extended nondividing cells that contain spiral FtsZ complexes. FtsZ was visualized by immunofluorescence. B. Model of FtsZ protein monomer based on X-ray crystallography shows the position of the mutant residues, D212A and D45A.
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FIGURE 3.5 ■ Bacterial cell membrane.
The cell membrane consists of a phospholipid bilayer, with hydrophobic fatty acid chains directed inward, away from water. The bilayer contains stiffening agents such as hopanoids. Half the membrane volume consists of proteins.
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LeuT sodium/leucine cotransporter
Homology to human neurotransmitter sodium sympoters
Has been used as a blueprint to understand structure and function, and pharmacology of NSS human transporters.
FIGURE 3.7 (part 1) ■ A cell membrane–embedded transport protein: the LeuT sodium/leucine cotransporter of Aquifex bacteria.
The protein complex carries leucine across the cell membrane into the cytoplasm, coupled to sodium ion influx. (PDB code: 3F3E)
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Transport across bacterial membranes
Passive diffusion
Membrane proteins
Aquaporins
Permease (lac operon)
Osmosis
Greater osmotic pressure can lead to bacterial cell lysis (seen with certain antibiotics)
Membrane-permeant weak acids and bases: can cross the plasma membrane
Transmembrane ion gradients
FIGURE 3.8 ■ Common drugs are membrane-permeant weak acids and bases.
In its charged form (A– or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.
*
FIGURE 3.8a ■ Common drugs are membrane-permeant weak acids and bases.
In its charged form (A– or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.
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FIGURE 3.8b ■ Common drugs are membrane-permeant weak acids and bases.
In its charged form (A– or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.
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NAM and NAG are linked together by a β-(1,4)-glycosidic bond
Lysozymes target this bond
The peptidoglycan monomer will have 5 peptides
Once this monomer becomes incorporated into the existing polymer, 4 peptides are seen.
FIGURE 3.14b ■ The peptidoglycan sacculus and peptidoglycan cross-bridge formation.
B. A disaccharide unit of glycan has an attached peptide of four to six amino acids.
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FIGURE 3.16 ■ Cell envelope: Gram-positive (Firmicutes) and Gram-negative (Proteobacteria).
A. Firmicutes (Gram-positive) cells have a thick cell wall with multiple layers of peptidoglycan, threaded by teichoic acids. A inset: Gram-positive envelope of Bacillus subtilis (TEM). B. Proteobacteria (Gram-negative) cells have a single layer of peptidoglycan covered by an outer membrane; the cell membrane is called the inner membrane. B inset: Gram-negative envelope of Pseudomonas aeruginosa (TEM).
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Gram +
FIGURE 3.19a ■ Gram-negative cell envelope.
A. Murein lipoprotein has an N-terminal cysteine triglyceride inserted in the inward-facing leaflet of the outer membrane. The C-terminal lysine forms a peptide bond with the m-diaminopimelic acid of the peptidoglycan (murein) cell wall.
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FIGURE 3.20 ■ Lipopolysaccharide (LPS).
A. Lipopolysaccharide (LPS) consists of core polysaccharide and O antigen linked to a lipid A. Lipid A consists of a dimer of phosphoglucosamine esterified or amidated to six fatty acids. B. Repeating polysaccharide units of O antigen extend from lipid A.
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FIGURE 3.20a ■ Lipopolysaccharide (LPS).
A. Lipopolysaccharide (LPS) consists of core polysaccharide and O antigen linked to a lipid A. Lipid A consists of a dimer of phosphoglucosamine esterified or amidated to six fatty acids.
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FIGURE 3.20b ■ Lipopolysaccharide (LPS).
B. Repeating polysaccharide units of O antigen extend from lipid A.
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4.3 Culturing and Counting Bacteria
Microbes in nature exist in complex, multispecies communities, but for detailed studies they must be grown separately in pure culture.
After
1
2
0 years of trying, we have succeeded in culturing less than 1% of the microorganisms around us.
The vast majority has yet to be tamed.
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Bacteria Are Grown in Culture Media – 1
Bacteria are grown in culture media, which are of two main types:
Liquid or broth
Useful for studying the growth characteristics of a pure culture
Solid (usually gelled with agar)
Useful for trying to separate mixed cultures from clinical specimens or natural environments
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Bacteria Are Grown in Culture Media – 2
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FIGURE 4.12 ■ Separation and growth of microbes on an agar surface. A. Colonies (diameter 1–5 mm) of Acidovorax citrulli separated on an agar plate. A. citrulli is a plant pathogen that causes watermelon fruit blotch. B. A mixture of yellow-pigmented bacterial colonies, wrinkled bacterial colonies, and fungus separated by dilution on an agar plate. As time passed, the fungal colony overgrew adjacent bacterial colonies.
Dilution Streaking and Spread Plates – 1
Pure colonies are isolated via two main techniques:
Dilution streaking
A loop is dragged across the surface of an agar plate.
Spread plate
Tenfold serial dilutions are performed on a liquid culture.
A small amount of each dilution is then plated.
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Dilution Streaking and Spread Plates – 2
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FIGURE 4.13 ■ Dilution streaking technique. A. A liquid culture is sampled with a sterile inoculating loop and streaked across the plate in three or four areas, with the loop flamed between areas to kill bacteria still clinging to it. Dragging the loop across the agar diminishes the number of organisms clinging to the loop until only single cells are deposited at a given location. B. Salmonella enterica culture obtained by dilution streaking.
Dilution Streaking and Spread Plates – 3
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FIGURE 4.15 ■ Tenfold dilutions, plating, and viable counts. A. A culture containing an unknown concentration of cells is serially diluted. One milliliter (ml) of culture is added to 9.0 ml of diluent broth and mixed, and then 1 ml of this 1/10 dilution is added to another 9.0 ml of diluent (10–2 dilution). These steps are repeated for further dilution, each of which lowers the cell number tenfold. After dilution, 0.1 ml of each dilution is spread onto an agar plate. B. Plates prepared as in (A) are incubated at 37°C to yield colonies. By multiplying the number of countable colonies (107 colonies on the 10–5 plate) by 10, you get the number of cells in 1.0 ml of the 10–5 dilution. Multiplying that number by the reciprocal of the dilution factor, you can calculate the number of cells (colony-forming units, or CFUs) per milliliter in the original broth tube (107 x 101 x 105 = 1.1 x 108 CFUs/ml). TNTC = too numerous to count.
Types of Media – 1
Complex media are nutrient rich but poorly defined.
Minimal defined media contains only those nutrients that are essential for growth of a given microbe.
Enriched media are complex media to which specific blood components are added.
Selective media favor the growth of one organism over another.
Differential media exploit differences between two species that grow equally well.
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Types of Media – 2
Medium Ingredients Amount per liter Organisms cultured
Luria Bertani (complex) Bacto tryptone, a pancreatic digest of casein (bovine milk protein) 10 grams Many Gram-negative and Gram-positive organisms (such as Escherichia coli and Staphylococcus aureus, respectively)
Luria Bertani (complex) Bacto yeast extract 5 grams Empty cell
Luria Bertani (complex) Upper N a upper C l Adjust to pH 7 10 grams Empty cell
M 9 medium (defined) Glucose 2.0 grams Gram-negative organisms such as E. coli
M 9 medium (defined) Upper N a 2 upper H upper P upper O 4 6.0 grams (42 millimolar) Gram-negative organisms such as E. coli
M 9 medium (defined) Upper K upper H 2 upper P upper O 4 3.0 grams (22 millimolar) Gram-negative organisms such as E. coli
M 9 medium (defined) Upper N upper H 4 upper C l 1.0 grams (19 millimolar) Gram-negative organisms such as E. coli
M 9 medium (defined) Upper N a upper C l 0.5 grams (9 millimolar) Gram-negative organisms such as E. coli
M 9 medium (defined) Upper M g upper S upper O 4 2.0 millimolar Gram-negative organisms such as E. coli
M 9 medium (defined) Upper C a upper C l 2 Adjust to pH 7 0.1 millimolar Gram-negative organisms such as E. coli
Sulfur oxidizers (defined) Upper N upper H 4 upper C l 0.52 grams Acidithiobacillus thiooxidans
Sulfur oxidizers (defined) Upper K upper H 2 upper P upper O 4 0.28 grams Acidithiobacillus thiooxidans
Sulfur oxidizers (defined) Upper M g upper S upper O 4 times 7 upper H 2 upper O 0.25 grams Acidithiobacillus thiooxidans
Sulfur oxidizers (defined) Upper C a upper C l 2 0.07 grams Acidithiobacillus thiooxidans
Sulfur oxidizers (defined) Elemental sulfur 1.56 grams Acidithiobacillus thiooxidans
Sulfur oxidizers (defined) Upper C upper O 2 Adjust to pH 3 5 percent Acidithiobacillus thiooxidans
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Types of Media – 3
Several media used in clinical microbiology are both selective and differential.
e.g., MacConkey medium
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FIGURE 4.16 ■ MacConkey medium, a culture medium both selective and differential. Only Gram-negative bacteria grow on lactose MacConkey (selective). Only a species capable of fermenting lactose produces pink colonies (differential), because only fermenters can take up the neutral red and peptones that are also in the medium. Gram-negative nonfermenters appear as uncolored colonies.
Growth Factors, Unculturable Microbes, and Obligate Intracellular Bacteria – 1
Microbes can evolve to require specific growth factors depending on the nutrient richness of their natural ecological niche.
Growth factors are specific nutrients not required by other species.
A microbe needs them in order to be able to grow in laboratory media.
e.g.: Streptococcus pyogenes requires glutamic acid and alanine because it can no longer synthesize them.
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Growth Factors, Unculturable Microbes, and Obligate Intracellular Bacteria – 2
Organism Diseases Natural habitats Growth factors
Shigella Bloody diarrhea Humans Nicotinamide, which is derived from upper N upper A upper D, nicotinamide adenine dinucleotide
Haemophilus Meningitis, chancroid Humans and other animal species, upper respiratory tract Hemin, upper N upper A upper D
Staphylococcus Boils, osteomyelitis Widespread Complex requirement
Abiotrophia Osteomyelitis Humans and other animal species Vitamin K, cysteine
Legionella Legionnaires’ disease Soil, refrigeration cooling towers Cysteine
Bordetella Whooping cough Humans and other animal species Glutamate, proline, cysteine
Francisella Tularemia Wild deer, rabbits Complex, cysteine
Mycobacterium Tuberculosis, leprosy Humans Nicotinic acid, which is derived from upper N upper A upper D, nicotinamide adenine dinucleotide, and alanine (M. leprae is unculturable)
Streptococcus pyogenes Pharyngitis, rheumatic fever Humans Glutamate, alanine
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Growth Factors, Unculturable Microbes, and Obligate Intracellular Bacteria – 3
Some species have adapted so well to their natural habitats that we still do not know how to grow them in the lab.
Some of these “unculturable” organisms depend on factors provided by other species that cohabit their niche.
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FIGURE 4.17 ■ “Unculturable” marine organism MSC33. To grow in natural environments, many bacterial species rely on factors produced by other species within their niche. The microbe shown, MSC33, will not grow in laboratory media unless a peptide growth factor from another species is included.
Growth Factors, Unculturable Microbes, and Obligate Intracellular Bacteria – 4
Obligate intracellular bacteria are also unculturable.
e.g.: Rickettsia prowazekii, the cause of epidemic typhus fever, has adapted to grow within the cytoplasm of eukaryotic cells and nowhere else.
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FIGURE 4.18 ■ Rickettsia prowazekii growing within eukaryotic cells. A. R. prowazekii growing within the cytoplasm of a chicken embryo fibroblast (SEM). B. Fluorescent stain of R. prowazekii (approx. 0.5 μm long), growing within a cultured human cell (outline marked by dotted line). The rickettsias are green (FITClabeled antibody, arrow), the host cell nucleus is blue (Hoechst stain), and the mitochondria are red (Texas Red MitoTracker). The bacterium grows only in the cytoplasm, not in the nucleus.
Techniques for Counting Bacteria
There are many reasons why it is important to know the number of organisms in a sample.
Counting or quantifying organisms invisible to the naked eye is surprisingly difficult.
Each of the available techniques measures a different physical or biochemical aspect of growth.
Thus, cell density values derived from these techniques may not necessarily agree with one another.
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Direct Counting of Living and Dead Cells – 1
Microorganisms can be counted directly by placing dilutions on a special microscope slide called a Petroff-Hausser counting chamber.
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FIGURE 4.19 ■ The Petroff-Hausser chamber for direct microscopic counts. A precision grid is etched on the surface of the slide. The organisms in several squares are counted, and their numbers are averaged. Knowing the dimensions of the grid and the height of the coverslip over the slide makes it possible to calculate the number of organisms in a milliliter.
Direct Counting of Living and Dead Cells – 2
Living cells may be distinguished from dead cells by fluorescence microscopy using fluorescent chemical dyes.
Dead bacterial cells fluoresce orange or yellow because propidium (red) can enter the cells and intercalate the base pairs of DNA.
Live cells fluoresce green because Syto-9 (green) enters the cell.
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FIGURE 4.20 ■ Live/dead stain. Live and dead bacteria visualized on freshly isolated human cheek epithelial cells using the LIVE/DEAD BacLight Bacterial Viability Kit. Dead bacterial cells fluoresce orange or yellow because propidium (red) can enter the cells and intercalate the base pairs of DNA. Live cells fluoresce green because Syto-9 (green) enters the cell. The faint green smears are the outlines of cheek cells.
Direct Counting of Living and Dead Cells – 3
Direct counting without microscopy can be done using an electronic technique that not only counts but also separates populations of bacterial cells according to their distinguishing properties.
The instrument is called a fluorescence-activated cell sorter (FACS) or flow cytometer.
Fluorescent cells are passed through a small orifice and then past a laser.
Detectors measure light scatter in the forward direction (measure of particle size) and to the side (particle shape or granularity).
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Direct Counting of Living and Dead Cells – 4
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FIGURE 4.21 ■ Fluorescence-activated cell sorter (FACS). A. Schematic of a FACS apparatus (bidirectional sorting). B. Separation of GFP-producing E. coli from non-GFP-producing E. coli. The low-level fluorescence in the cells on the left is baseline fluorescence (autofluorescence). The scatterplot displays the same FACS data, showing the size distribution of cells (x-axis) with respect to the level of fluorescence (y-axis). The larger cells may be cells that are about to divide.
Other Techniques
A viable bacterium is defined as being capable of replicating and forming a colony on a solid medium.
Viable cells can be counted via the pour plate method.
Microorganisms can be counted indirectly via biochemical assays of cell mass, protein content, or metabolic rate.
Also by measuring optical density
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4.4 The Growth Cycle
Most bacteria divide by binary fission, where one parent cell splits into two equal daughter cells.
However, some divide asymmetrically.
Hyphomicrobium divides by budding.
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FIGURE 4.22 ■ Symmetrical and asymmetrical cell division. A. Symmetrical cell division, or binary fission, in Lactobacillus sp. (SEM). B. Asymmetrical cell division via budding in the marine bacterium Hyphomicrobium (approx. 4 μm long).
Exponential Growth
The growth rate, or rate of increase in cell numbers or biomass, is proportional to the population size at a given time.
Such a growth rate is called “exponential” because it generates an exponential curve, a curve whose slope increases continually.
If a cell divides by binary fission, the number of cells is proportional to 2n.
Where n = number of generations
Note: Some cyanobacteria divide by multiple fission.
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Generation Time
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Stages of Growth in a Batch Culture
Exponential growth never lasts indefinitely.
The simplest way to model the effects of a changing environment is to culture bacteria in a batch culture.
A liquid medium within a closed system
The changing conditions in this system greatly affect bacterial physiology and growth.
This illustrates the remarkable ability of bacteria to adapt to their environment.
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Bacterial Growth Curves
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FIGURE 4.23 ■ Bacterial growth curves. A. Theoretical growth curve of a bacterial suspension measured by optical density (OD) at a wavelength of 600 nm. B. Phases of bacterial growth in a typical batch culture.
Continuous Culture – 1
In a continuous culture, all cells in a population achieve a steady state, which allows detailed study of bacterial physiology.
The chemostat ensures logarithmic growth by constantly adding and removing equal amounts of culture media.
Note that the human gastrointestinal tract is engineered much like a chemostat.
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Continuous Culture – 2
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FIGURE 4.25 ■ Chemostats and continuous culture. A. The basic chemostat ensures logarithmic growth by constantly adding and removing equal amounts of culture media. B. The human gastrointestinal tract is engineered much like a chemostat, in that new nutrients are always arriving from the throat while equal amounts of bacterial culture exit in fecal waste. C. A modern chemostat.
Continuous Culture – 3
The complex relationships among dilution rate, cell mass, and generation time in a chemostat are illustrated here.
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FIGURE 4.26 ■ Relationships among chemostat dilution rate, cell mass, and generation time. As the dilution rate (x-axis) increases, the generation time decreases and the mass of the culture increases. When the rate of dilution exceeds the division rate, cells are washed from the vessel faster than they can be replaced by division, and the cell mass decreases. The y-axis varies depending on the curve, as labeled.
4.5 Biofilms – 1
In nature, many bacteria form specialized, surface-attached communities called biofilms.
These can be constructed by one or multiple species and can form on a range of organic or inorganic surfaces.
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FIGURE 4.27 ■ Biofilms. A. A greenish-brown slime biofilm found on cobbles of the streambed in High Ore Creek, Montana. B. The biofilm that forms on teeth is called plaque.
4.5 Biofilms – 2
Bacterial biofilms form when nutrients are plentiful.
Once nutrients become scarce, individuals detach from the community to forage for new sources of nutrients.
Biofilms in nature can take many different forms and serve different functions for different species.
The formation of biofilms can be cued by different environmental signals in different species.
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4.5 Biofilms – 3
Chemical signals enable bacteria to communicate (quorum sensing) and in some cases to form biofilms.
Biofilm development involves:
The adherence of cells to a substrate
The formation of microcolonies
Ultimately, the formation of complex channeled communities that generate new planktonic cells
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Biofilm Development – 1
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FIGURE 4.28 ■ Biofilm development. The stages of biofilm development in Pseudomonas, which generally apply to the formation of many kinds of biofilms. Inset: A mucoid environmental strain of P. aeruginosa produces uneven, lumpy biofilms in an experimental flow cell (see Fig. 2.18). Cells in the biofilm were stained green with the fluorescent DNAbinding dye Syto-9 (3D confocal laser scanning microscopy). Source: H. C. Flemming and J. Wingender. 2010. Nat. Rev. Microbiol. 8:623–633.
Biofilm Development – 2
For many bacteria, sessile (nonmoving) cells in a biofilm chemically “talk” to each other in order to build microcolonies and keep water channels open.
Bacillus subtilis also spins out a fibril-like amyloid protein called TasA, which tethers cells and strengthens biofilms.
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FIGURE 4.29 ■ Floating biofilm (pellicle) formation of Bacillus subtilis. Cells were grown in a broth for 48 hours without agitation at 30°C. The pellicles formed by wild-type and tasA mutant B. subtilis are strikingly different. Wild-type pellicles are extremely wrinkly (A), whereas tasA mutant pellicles are flat and fragile (B). Insets: Electron micrographs of wild-type (A) and tasA mutant (B) cells.
Background
Is a non-pathogenic bacterium, which is the model organism for studying the formation and growth of bacterial biofilms.
B. subtilus is non-pathogenic, which means does not cause disease.
Several bacterial species, prefer to live under B. subtilus biofilms, as they are robust, and provide a barrier of protection from environmental stressors (chemicals, or protection from other microorganisms).
Pathogenic (disease causing) bacteria in the wild primarily prefer B. subtilus biofilms.
An example of a pathogen frequently found in B. subtilus biofilms is Bacillus cereus.
In the same genus as Bacillus
A well known food poisoning bacterium
Forms its own biofilm, but when grown together with B. subtilus, will send signals to have B. subtilus create the biofilm.
Bacillus subtilus
Bacillus cereus
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B. subtilus biofilm formation is possible by adding 1% glycerol/0.1mM MnSO4 to LB media
LB only
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Biofilm composition in B. subtilus and B. cereus
B. subtilus and B. cereus share homology between genes of the epsA-epsO and tapA operons that are involved in biofim formation.
The epsA-epsO is an operon composed of 15 genes, which encode proteins responsible for the formation of a exopolysaccharide (sugars) in the extracellular matrix that hold bacteria (not only B. subtilus and B. cereus) in these biofilms together.
The tap A operon contains a gene known as tasA, which encodes a protein that produces amyloid fibers.
The epsA-epsO and tapA are inducible operons, meaning the genes are not expressed until needed.
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Triggers of biofilm formation in B. subtilus
Biofilm formation requires specific nutrients in order to create the exopolysaccharide (EPS), and amyloid fibers.
The two nutrients needed by B. subtilus and B. cereus to create biofilms are glycerol (C3H8O3), and manganese (Mn++).
The picture on the right is a depiction of a signal transduction pathway in B. subtilus and B. cereus, which ultimately leads to the induction of the epsA-epsO and tapA operons.
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1% Glycerol/0.1mM MnSO4 is necessary and sufficient for complete biofilm formation
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